S. pneumoniae is an important agent of disease in humans, especially among infants, the elderly and immunocompromised persons. It is a bacterium frequently isolated from patients with invasive diseases such as bacteraemia/septicaemia, pneumonia, and meningitis with high morbidity and mortality throughout the world. Although the advent of antimicrobial drugs has reduced the overall mortality from pneumococcal diseases, the presence of resistant pneumococcal organisms has become a major problem in the world today. Effective pneumococcal vaccines could have a major impact on the morbidity and mortality associated with S. pneumoniae disease. Such vaccines would also potentially be useful to prevent otitis media in infants and young children.
It is clear that a number of pneumococcal factors are potentially important in the pathogenesis of disease G. J. Boulnois, J. Gen. Microbiol., 138, pp. 249-259 (1992); C. J. Lee et al., Crit. Rev. Microbiol., 18, pp. 89-114 (1991)!. The capsule of the pneumococcus, despite its lack of toxicity, is considered to be the sine qua non of pneumococcal virulence. More than 80 pneumococcal capsular serotypes are identified on the basis of antigenic differences. Antibodies are the mechanism of protection and the importance of anticapsular antibodies in host defenses against S. pneumoniae is well established R. Austrian, Am. J. Med., 67, pp. 547-549 (1979)!. Nevertheless, the currently available pneumococcal vaccine, comprising 23 capsular polysaccharides that most frequently caused disease, has significant shortcomings such as the poor immunogenicity of capsular polysaccharides, the diversity of the serotypes and the differences in the distribution of serotypes over time, geographic areas and age groups. In particular, the failure of existing vaccines to protect young children against most serotypes has spurred evaluation of other S. pneumoniae components. Increasing evidence indicates that certain pneumococcal proteins may play an active role both in terms of protection and pathogenicity J. C. Paton, Ann. Rev. Microbiol., 47, pp. 89-115 (1993)!. So far, however, only a few S. pneumoniae proteins have been studied. This might result from the lack of proteinspecific antibodies which renders difficult the study of the role of protein antigens in protection and pathogenicity. It is believed that the pneumococcal protein antigens are not very immunogenic and that most antibody responses are to the phosphocholine and the capsular polysaccharides L. S. McDaniel et al., J. Exp. Med., 160, pp. 386-397 (1984); R. M. Krause, Adv. Immunol., 12, pp. 1-56 (1970); D. G. Braun et al., J. Exp. Med., 129, pp. 809-830 (1969)!. In a study using X-linked immunodeficient mice, which respond poorly to carbohydrate antigens and to phosphocholine, but make relatively normal responses to protein antigens, the frequency for obtaining monoclonal antibodies reactive with pneumococcal protein antigens was less than 10%, thus suggesting that S. pneumoniae proteins are poor immunogens McDaniel et al., supra!.
Heat shock or stress proteins ("HSPs") are among the most highly conserved and abundant proteins found in nature F. C. Neidhardt et al., Ann. Rev. Genet., 18, pp. 295-329 (1984); S. Lindquist, Ann. Rev. Biochem., 55, pp. 1151-1191 (1986)!. They are produced by all cells in response to various physiological and nonphysiological stimuli. The heat shock response, in which a sudden increase in temperature induces the synthesis of HSPs, is the best studied of the stress responses. Other environmental conditions such as low pH, iron deficiency and hydrogen peroxyde can also induce HSPs. The HSPs have been defined by their size, and members of hsp90, hsp70, and hsp6o families are among the major HSPs found in all prokaryotes and eukaryotes. These proteins fulfill a variety of chaperon functions by aiding protein folding and assembly and assisting translocation across membranes C. Georgopoulos and W. J. Welch, Ann. Rev. Cell. Biol., 9, pp. 601-634 (1993); D. Ang et al., J. Biol. Chem., 266, pp. 24233-24236 (1991)!. As molecular chaperons and possibly via other mechanisms, HSPs are likely involved in protecting cells from the deleterious effects of stress. The fact that several virulence factors are regulated by environmental conditions suggests a role for HSPs in microbial pathogenicity J. J. Mekalanos, J. Bacteriol., 174, pp. 1-7 (1992); P. J. Murray and R. A. Young, J. Bacteriol., 174, pp. 4193-4196 (1992)!. In that respect, recent studies on Salmonella species suggest that the stress response might be critically linked to the ability of intracellular pathogens to initiate and sustain an infection N. A. Buchmeir and F. Heffron, Science, 248, pp. 730-732 (1990); K. Z. Abshire and F. C. Neidhardt, J. Bacteriol., 175, pp. 3734-3743 (1993); B. B. Finlay et al., Science, 243, pp. 940-943 (1989)!. Others have demonstrated that lysteriolysin, an essential virulence factor in L. monocytogenes, is induced under heat shock conditions Z. Sokolovic and W. Goebel, Infect. Immun., 57, pp. 295-298 (1989)!.
Evidence is now accumulating that HSPs are major antigens of many pathogens. Members of the hsp60 family, also called GroEL-related proteins for their similarity to the E. coli GroEL protein, are major antigens of a variety of bacterial pathogens including Mycobacterium leprae and Mycobacterium tuberculosis D. Young et al., Proc. Natl. Acad. Sci. USA, 85, pp. 4267-4270 (1988)!, Legionella pneumophila B. B. Plikaytis et al., J. Clin. Microbiol., 25, pp. 2080-2084 (1987)!, Borrelia burgdorferi B. J. Luft et al., J. Immunol., 146, pp. 2776-2782 (1991)!, and Chlamydia trachomatis E. A. Wagar et al., J. Infect. Dis., 162, pp. 922-927 (1990)!. This antigen is a homolog of the ubiquitous "common antigen", and is believed to be present in every bacterium J. E. Thole et al., Microb. Pathogen., 4, pp. 71-83 (1988). Antibodies to the members of the hsp70 family, or DnaK-related proteins, have also been described for several bacterial and parasitic infections Young et al., supra; Luft et al., supra; D. M. Engman et al., J. Immunol., 144, pp. 3987-3991 (1990); N. M. Rothstein et al., Molec. Biochem. Parasitol., 33, pp. 229-235 (1989); V. Nussenzweig and R. S. Nussenzweig, Adv. Immunol., 45, pp. 283-334 (1989)!. HSPs can elicit strong B- and T- cell responses and it was shown that 20% of the CD4.sup.+ T-lymphocytes from mice inoculated with M. tuberculosis were reactive to the hsp60 protein alone S. H .E. Kaufman et al., Eur. J. Immunol., 17, pp. 351-357 (1987)!. Similarly, 7 out of a collection of 24 monoclonal antibodies to M. leprae proteins recognized determinants on hsp60 H. D. Engers et al., Infect. Immun., 48, pp. 603-605 (1985)!. It seems that the immune response to stress proteins might play an important role in protection against infection. Consistent with that is the demonstration that antibodies and T cells reactive with microbial HSPs can exhibit neutralizing and protective activities A. Noll et al., Infect. Immun., 62, pp. 2784-2791 (1994); and S. L. Danilition et al., Infect. Immun., 58, pp. 189-196 (1990)!. The immunological properties of stress proteins make them attractive as vaccine components and several HSPs are presently being considered for preventing microbial infection and treating cancer. So far, however, studies have focused on intracellular pathogens such as Mycobacteria, Salmonella, Chlamydia and several parasites. Information concerning the heat shock protein antigens in extracellular gram-positive bacteria is far less documented. In S. pneumoniae, neither the heat shock proteins nor their gene structure has been identified.